Production of poxviruses with adherent or non adherent avian cell lines

ABSTRACT

The present invention relates to a method for replicating pox viruses such as vaccinia virus comprising the steps of inoculating avian embryonic stem cells with viral particles and culturing said cells in a basal medium until cells lysis occurs and newly produced viral particles are released in said medium.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. national stage of International ApplicationNo. PCT/IB2004/002621, filed Jul. 22, 2004, incorporated by referenceherein in its entirety and relied upon, which claims priority under 35U.S.C. §119 of Application No. 03291813.8, filed in the European PatentOffice on Jul. 22, 2003 and Application No. 0314389, filed in France onDec. 9, 2003.

The present invention relates to a method for producing live orattenuated pox viruses, in particular vaccinia viruses, native ormodified, with avian cell lines, in particular avian embryonic stemcells, comprising infecting said cells with virus particles.

Historically, vaccinia virus is known to have been used successfully toimmunize against smallpox allowing its eradication in 1980 according tothe WHO. Since then, vaccination has been discontinued. Today,resurgence of this virus is considered as a potential threat that couldbe devastating for the unprotected population. The problem is that only15 million doses of smallpox vaccine are available in the USA and theFDA has issued guidelines and contracts to produce large amounts ofvaccine unit dose against smallpox. Further information are accessibleat:

http://www.bt.cdc.gov/Agent/Smallpox/SmallpoxConsensus.pdf.

However, speeding-up production requires new production methods andsuitable cell lines. In this invention, we describe new cell lines(adherent or non adherent) from avian species that could be used assubstrate for vaccine production by pharmaceutical companies. These newcells are derived from avian embryos and could supersede eggs or primaryembryo fibroblasts that are used at present.

Traditionally, vaccines induce immunity to diseases by using a weakenedor inactivated version of the infectious agent. Today attenuated poxviruses properties like the absence of replication in human cells andthe good induction of immune response allow the development of newvaccine strategies using for example MVA (Modified Virus Ankara) asvector. MVA is a highly attenuated strain of vaccinia virus (VV) thatwas initially developed as a safe vaccine for smallpox prior toeradication of that disease. MVA was derived from the Ankara strain byover 570 serial passages through primary chick embryo fibroblasts (CEF)and, as a consequence of this adaptation, contains several large genomicdeletions compared to the parental strain. MVA can no longer replicatein most mammalian cell lines and is non pathogenic in animals. Moreimportantly, no serious complications were reported when MVA wasadministered as a smallpox vaccine in more than 100,000 humans,including immuno-depressed individuals. By classical techniques ofmolecular biology, it is possible to obtain recombinant MVA containingforeign DNA coding for specific peptides or proteins of therapeuticinterest. These recombinant viruses after injection, in vivo, are ableto stimulate the immune system against specific antigens like tumoralantigens. At present, these new generations of vaccine vectors aredeveloped or could be developed to fight against human or animalinfectious diseases and against a wide variety of tumor types (melanoma,prostate cancer, breast cancer, lung cancer, ovary cancer, liver cancer. . . ).

Drexler I. et al. (1998 J. Gen Virol. 79:347-352) have observed thathighly attenuated Modified Vaccinia virus Ankara (MVA) replicates inbaby hamster kidney cells (a potential host for virus propagation) butnot in various human transformed and primary cells; therefore, the hostrange of MVA is restricted. Moreover this highly attenuated poxvirusstrain do not create productive infections (Moss B, Dev Biol Stand1994:55-63). For example, Blanchard T J. et al. (1998 J. Gen. Virol.79:1159-1167) have reported that Modified Vaccinia virus Ankaraundergoes limited replication in human cells and lacks severalimmuno-modulatory proteins. In addition, production yields must becommensurate with economical viability of smallpox vaccine massproduction.

The strong safety record of MVA and the potent cellular and humoralimmune response elicited in vaccinated individuals has generatedconsiderable scientific and industrial interest in the use of MVA as arecombinant vector to immunize against human and animal infectiousdiseases, in particular against HIV and cancer. Given its adaptation toCEF, MVA can be grown to high titers in such cells and current clinicalbatches of recombinant MVA vaccine candidates are produced on primaryCEF. However, the establishment of CEFs requires experience in preparingprimary tissue culture and depends on eggs from chicken kept underspecial pathogen-free conditions. Furthermore, the production process isenormously laborious and difficult to standardize, since primary cellssurvive only a few passages and have thus to be prepared continuouslyfrom embryonated eggs. While vaccine producers can deal with suchlimitations for the production of batches of MVA material for phase Iand II clinical trials, scaling-up the CEF-based production process forphase III clinical trials and eventually for later productcommercialization remains a serious hurdle.

The problem to be solved by the present invention is to provide celllines for replicating the vaccinia virus which would obviate the abovementioned problems and which would meet with regulatory agencyrequirements. That's the purpose of the present invention.

Ideally, such a cell line should be fully regulatory compliant; the cellshall be fully characterized with a known history. Furthermore, the cellline shall be non tumorigenic, genetically unmodified, and stable underlong-term culture. The cell line shall be able to replicate viruses andadapted to stable adherent and suspension growth in serum-free medium.In this regards, the inventors investigated the use of avian cells forreplicating viruses. The inventors report that the established new avianembryonic derived stem cell lines detailed in our co-pending applicationPCT/FR03/00735 (WO03/076601), are particularly suitable for replicatingpoxviruses, in particular orthopoxvirus such as the vaccinia virus.

Because unlimited cell proliferation is required for the process ofvaccine mass production, the inventors choose to examine the avianembryonic derived stem cell ability for replicating viruses. However, tomaintain avian embryonic derived stem cells in vitro for long periods oftime, it is necessary to observe specific culture and maintenanceconditions as described in Pain et al. (1996, Development 122:2339-2348); U.S. Pat. No. 6,114,168 and EP 787 180 and these cultureconditions are cost demanding. The problem was to be able to maintainavian embryonic derived stem cells in culture in an economical mediumwhile avoiding stumbling blocks such as cellular differentiation andsenescence. In the context of the invention, it has been found that thewithdrawal of growth factors, serum and/or feeder layer leads to theisolation of populations of avian embryonic derived stem cells, whichcan grow indefinitely in basic culture media.

Also, apart from the hematopoietic stem cells which are for the mostpart non-adherent cells, the cells obtained according to the prior arttechniques exhibit an adherent phenotype. However, non-adherent cellsare preferred for the industrial production of viral vaccines. Thisphenotype is advantageous both because of ease of handling which avoidsthe use of a proteolytic enzyme for dissociation and for the high celldensities reached by non-adherent cells cultured in vitro. The presentinvention describes the production of avian embryonic derived stem celllines which can become spontaneously non-adherent or for which thenon-adherence is obtained by a withdrawal of the feeder layer. Becauseof their growth in suspension, these lines are perfectly suitable forindustrial production of vaccines in bioreactors.

In addition to their properties of growing on a basic culture medium, ithas been discovered that these cell lines allow the replication ofcertain viruses in yields equivalent to or even higher than yieldsobtained with current methods, which makes these cells particularlyuseful for the mass production of vaccines.

DESCRIPTION

Thus, in a first aspect, the present invention relates to a method forreplicating viruses, and more particularly vaccinia virus, such asnative or recombinant vaccinia virus in avian embryonic derived stemcells. The method of the invention comprises the steps of inoculatingsaid avian embryonic derived stem cells with viral particles andculturing said cells in a medium deprived in growth factors, feedercells and/or animal serum, until cells lysis occurs and newly producedviral particles are released in said medium. Inoculation of avian stemcells of the invention is performed with an m.o.i. (multiplicity ofinfection) of 0.001 to 0.5, in a preferred embodiment of 0.01 to 0.5 andin a most preferred embodiment 0.01 to 0.1. This method is useful forproducing vaccines, specially vaccines against poxviridae in particularagainst smallpox.

Said avian embryonic derived stem cell lines are obtainable by a processconsisting of:

-   -   a) culturing avian cells, preferably avian embryonic, in a        complete culture medium containing all the factors allowing        their growth and a feeder layer, preferably inactivated, and        complemented in serum;    -   b) passage by modifying the culture medium so as to obtain        progressive or total withdrawal of said factors, of the serum        and/or of the feeder layer,    -   c) establishing adherent or non adherent avian cell lines        capable of proliferating in a basal medium in the absence of        exogenous growth factors, and/or inactivated feeder layer and/or        a low level of serum or no serum;        In the event, the basal medium of step c) still comprises a low        level of serum (i.e. around 2% or less), said process may        optionally comprises an additional step d) of changing the basal        medium containing no more exogenous growth factor, no more        inactivated feeder layer and a low level of serum in a medium of        culture selected among:    -   a basal medium complemented with serum (i) and diluted with a        serum-free medium, then culturing during successive passages        said avian cells in the basal medium (i) in which the ratio of        serum-free medium is progressively increased up to the complete        disappearance of said basal medium containing no exogenous        growth factor, no inactivated feeder layer and a low level of        serum;    -   a serum-free medium (SFM) complemented with serum (ii), then        culturing during successive passages said avian cells in said        medium (ii) in which the ratio of serum is progressively        decreased up to the obtaining of a serum-free medium;    -   a serum-free medium (SFM) (iii), then culturing said avian cells        in medium (iii); then maintaining in serum-free medium said        avian cells adapted to the medium change.

The term <<avian>> as used herein is intended to refer to any species,subspecies or race of organism of the taxonomic class <<ava>>, such as,but not limited to, such organisms as chicken, turkey, duck, goose,quails, pheasants, parrots, finches, hawks, crows, ostrich, emu andcassowary. The term includes the various strains of Gallus gallus, orchickens (for example White Leghorn, Brown Leghorn, Barred-Rock, Sussex,New Hampshire, Rhode Island, Ausstralorp, Minorca, Amrox, CaliforniaGray, Italian Partidge-colored), as well as strains of turkeys,pheasants, quails, duck, ostriches and other poultry commonly bred. In apreferred embodiment, the avian cell of the present invention is achicken cell.

The term “factor allowing their growth” as used herein meant growthfactor necessary for the survival and the growth of the avian cells inculture. According to the invention, the growth factors comprisestrophic factors and cytokines. Trophic factors are mainly SCF, IGF-1 andbFGF. Cytokines are mainly cytokines whose action is through a receptorwhich is associated with the gp130 protein such as LIF, interleukin 11,interleukin 6, interleukin 6 receptor, CNTF, oncostatin andcardiotrophin.

The avian cells of step a) are cells selected among avian embryoniccells, more preferably among avian embryonic stem cells and avianprimary cells. In a preferred embodiment, the cells are totipotent orpluripotent avian embryonic stem cells isolated from a populationsuspension of dissociated stage X blastodermal cells obtained from anavian embryo, more preferably a chicken embryo (see EYAL-GILADI'sclassification: EYAL-GILADI and KOCHAN, 1976, <<From cleavage toprimitive streack formation: a complementary normal table and a new lookat the first stages of the development in the chick>>. “GeneralMorphology” Dev. Biol. 49: 321-337). These avian embryonic stem cellsare characterized by a slow doubling time comprises between 48 to 72hours in culture at 39° C.

The modification of the culture medium of step b) of the process of theinvention, so as to obtain progressive or total withdrawal of growthfactors, serum and/or feeder layer, can be made simultaneously,successively or separately. The sequence of the weaning of the culturemedium may be chosen among:

-   -   feeder layer/serum/growth factors;    -   feeder layer/growth factors/serum;    -   serum/growth factors/feeder layer;    -   serum/feeder layer/growth factors;    -   growth factors/serum/feeder layer,    -   growth factors/feeder layer/serum.        In a preferred embodiment, the sequence of the weaning is growth        factors/feeder layer/serum.

In a particular embodiment, the invention relates to a method as definedabove, in which the established lines are adherent stem cells whichproliferate in the absence of inactivated feeder layer. In this regard,in the method described above, step b) consists in a withdrawal of thecomponents of the medium (growth factors alone or serum alone or growthfactors and then serum or alternatively serum and then growth factors).

In another embodiment, the invention relates to a method as definedabove in which the established lines are non adherent stem cells whichproliferate in suspension in a medium free of exogenous growth factors.In this regard, in the method described above, step b) consists in aprogressive or total withdrawal of the feeder layer and then optionallyin a withdrawal of the other components of the medium (growth factorsand serum).

In another embodiment, the invention relates to a method as describedabove in which the established lines are non adherent stem cells whichproliferate in suspension in a medium free of serum (serum-free medium).

In another embodiment, the invention relates to a method as definedabove, in which the established lines are non adherent stem cells whichproliferate in suspension in a medium free of exogenous growth factorsand serum.

In another alternative, step b) consists in a progressive or totalwithdrawal of the growth factors, optionally followed by a progressivewithdrawal of the serum.

In another alternative, step b) consists in a progressive or totalwithdrawal of the growth factors and/or serum, optionally followed by awithdrawal of the feeder layer.

In addition, the established lines may be cells which proliferate in aserum-depleted medium, in particular in a medium free of serum. Theexpression serum-depleted is understood to mean a gradual reduction ofthe concentration of serum spread out over time. This method allows aselection of clones which adapt to these new, increasingly drasticconditions until stable lines are obtained which are capable of growingin a serum-depleted medium or in a medium completely free of serum.

More precisely, step a) of the process comprises the seeding of cultureflasks with around between 7×10⁴/cm² to 8×10⁴/cm² avian cells in acomplete culture medium. Preferably, the seeding is made with around7.3×10⁴/cm² (4×10⁶ cells/55 cm² or 4×10⁶ cells/100 mm dish).

By “complete culture medium”, it is meant a basal medium complementedwith growth factors and animal serum. Example of complete culture mediumis described in Pain et al. (1996, Development 122:2339-2348), EP787,180and U.S. Pat. No. 6,114,168, 5,340,740, 6,656,479 and 5,830,510.According to the invention, “basal medium” meant a medium with aclassical media formulation that allows, by itself, at least cellssurvival, and even better, cell growth. Examples of basal media are BME(basal EagleMedium), MEM (minimum Eagle Medium), medium 199, DMEM(Dulbecco's modified Eagle Medium), GMEM (Glasgow modified Eaglemedium), DMEM-HamF12, Ham-F12 and Ham-F10, Iscove's Modified Dulbecco'smedium, MacCoy's 5A medium, RPMI 1640. Basal medium comprises inorganicsalts (for examples: CaCl₂, KCl, NaCl, NaHCO₃, NaH₂PO₄, MgSO₄, . . . ),aminoacids, vitamins (thiamine, riboflavin, folic acid, D-Capanthothenate, . . . ) and others components such as glucose,beta-mercaptoethanol, sodium pyruvate.

It is possible to schematically distinguish two families of growthfactors: the cytokines and the trophic factors. The cytokines are mainlycytokines whose action is through a receptor which is associated withthe gp130 protein. Thus, LIF, interleukin 11, interleukin 6, interleukin6 receptor, CNTF, oncostatin and cardiotrophin have a similar mode ofaction with the recruitment at the level of the receptor of a specificchain and the combination of the latter with the gp130 protein inmonomeric or sometimes heterodimeric form. The trophic factors aremainly SCF, IGF-1 and bFGF. More preferably, the complete mediumcomprises basal medium, Insulin Growth factor 1 (IGF-1), CiliaryNeurotrophic factor (CNTF), Interleukine 6 (IL-6), interleukine 6receptor (IL-6R), Stem cell Factor (SCF), basic Fibroblast Growth Factor(bFGF), optionally interleukine 11 (IL-11) and animal serum. The aviancells, preferably the avian embryonic cells of step a) are culturedduring several passages in the complete medium. The medium iscomplemented by at least one of the growth factors selected in the groupof: LIF, IGF-1, CNTF, IL-6, IL-6R, SCF, bFGF, IL-11, oncostatin,cardiotrophin.

According to a preferred embodiment, the complete culture medium isbasal medium complemented with IGF-1, CNTF, IL-6, IL-6R, SCF, bFGF,optionally IL-11. The concentration of growth factors IGF-1, CNTF, IL-6,IL-6R, SCF, bFGF, optionally IL-11 in the basal medium is comprisedbetween about 0.01 to 10 ng/ml, preferably, 0.1 to 5 ng/ml, and morepreferably about 1 ng/ml.

After around passages 3 to 10, the complete medium is depleted in growthfactors (step b). Preferably, for each growth factor, the depletion ismade directly in one step, from one passage to another. Alternatively,the growth factor depletion is performed gradually, by a progressivedecrease of the growth factor concentration in the complete medium. In amore preferred embodiment, the growth factors depletion is performedsimultaneously for at least two growth factors. In a preferredembodiment, the depletion in growth factors is made in two rounds ofdepletion: firstly, SCF, IL6, IL6R, optionally IL11 are directly removedfrom the complete medium; the avian cells are then maintained in culturefor at least one passage in a complete medium containing IGF1 and CNTF,optionally IL-11, and supplemented with animal serum. Secondly, IGF1 andCNTF, optionally IL-11 are directly removed from the culture medium,which ultimately comprises the basal medium only supplemented withserum. Usually, the medium is totally depleted in growth factors ataround passages 20 to 30.

In a preferred embodiment, the deprivation of feeder cells is performedafter the deprivation of growth factors. The deprivation of feeder cellsis progressive and performed over several passages. The avian cells arenow seeded in flask at a lower concentration than in step a), aboutaround 4×10⁴ cell/cm² to 5×10⁴ cell/cm². The feeder cells are seeded inflask at around 4.2×10⁴ cell/cm². Progressively, the concentration ofthe feeder cells in the flask is decreased. Practically, the sameconcentration of the feeder cells is used for 2 to 4 passages, then alower concentration of the feeder cells is used for an additional 2 to 4passages, and so. The flask is then seeded with around 4.2×10⁴ feedercells/cm², then around 2.2×10⁴ feeder cells/cm², then around 1.8×10⁴feeder cells/cm², then around 1.4×10⁴ feeder cells/cm², then around1.1×10⁴ feeder cells/cm², then around 0.9×10⁴ feeder cells/cm², thenaround 0.5×10⁴ feeder cells/cm². Then the flask is seeded with 6.5×10⁴avian cells/cm² to 7.5×10⁴ avian cells/cm² and without feeder cells. Inthe hypothesis that avian cells are not in good shape following adecrease of feeder cells concentration in the flask, then the aviancells are cultured for additional passages with the same feeder cellsconcentration before to pursue the feeder cells deprivation.

In another preferred embodiment, the serum deprivation is performedafter the growth factor and the feeder cells deprivation. The basalmedium is changed by a medium selected among:

-   -   The basal medium (i) complemented with serum and diluted with a        novel serum free medium (ii). Then the avian cells are cultured        through successive passages in the medium (i) in which the serum        free medium proportion is progressively increased up to the        complete disappearing of the basal medium complemented in serum        (progressive dilution).    -   A novel serum free medium (ii) complemented with serum. Then the        avian cells are cultured through successive passages in the        medium (ii) in which the serum proportion is progressively        decreased up to the obtaining of a serum-free medium        (progressive weaning).    -   A novel serum free medium (ii) non complemented with serum. Then        the avian cells are directly in the serum-free medium (ii)        (direct weaning).        In a preferred embodiment, the serum deprivation is performed by        progressive weaning.

In a first embodiment, the method of serum deprivation proce

According to the present invention, “serum-free medium” (SFM) meant acell culture medium ready to use, that is to say that it does notrequired serum addition allowing cells survival and cell growth. Thismedium is not necessary chemically defined, and may containedhydrolyzates of various origin, from plant for instance. Preferably,said SFM are “non animal origin” qualified, that is to say that it doesnot contain components of animal or human origin (FAO status: “free ofanimal origin”). In SFM, the native serum proteins are replaced byrecombinant proteins. Alternatively SFM medium according to theinvention does not contain protein (PF medium: “protein free medium”)and/or are chemically defined (CDM medium: “chemically defined medium”).SFM media present several advantages: (i) the first of all being theregulatory compliance of such media (indeed there is no risk ofcontamination by adventitious agents such as BSE, viruses); (ii) theoptimization of the purification process; (iii) the betterreproducibility in the process because of the better defined medium.Example of commercially available SFM media are: VP SFM (InVitrogen Ref11681-020, catalogue 2003), Opti Pro (InVitrogen Ref 12309-019,catalogue 2003), Episerf (InVitrogen Ref 10732-022, catalogue 2003), Pro293 S-CDM (Cambrex ref 12765Q, catalogue 2003), LC17 (Cambrex RefBESP302Q), Pro CHO 5-CDM (Cambrex ref 12-766Q, catalogue 2003), HyQSFM4CHO (Hyclone Ref SH30515-02), HyQ SFM4CHO-Utility (Hyclone RefSH30516.02), HyQ PF293 (Hyclone ref SH30356.02), HyQ PF Vero (HycloneRef SH30352.02), Ex cell 293 medium (JRH Biosciences ref 14570-1000M),Ex cell 325 PF CHO Protein free medium (JRH Biosciences ref14335-1000M), Ex cell VPRO medium (JRH Biosciences ref 14560-1000M), Excell 302 serum free medium (JRH Biosciences ref 14312-1000M).

The invention also relates to a process of obtaining avian cell lines,preferably non transformed cell lines, able to grow in serum-freemedium; those cell lines are cultured in a complete culture mediumoptionally with feeder cells. Said process comprises the steps of:

-   -   culturing the avian cell, preferably non-transformed, in a        complete culture medium and optionally with feeder layer. The        avian cell may be the avian cells of step a) above, the        established avian cell lines of the process of the invention,        such as EB1, EB14 or S86N45 (also named EB45), or other avian        embryonic derived cell line such as DF1 (U.S. Pat. Nos.        5,672,485 and 6,207,415);    -   at least one passage in culture by modifying or changing the        culture medium in order to obtain a total weaning of serum,        either by progressive or direct withdrawal of serum;    -   establishing adherent or non-adherent avian cell lines able to        grow in serum-free medium.

The instant invention relies on the finding that the passage from abasal cell culture medium complemented with animal serum to a serum-freemedium shall not be performed by the simple removal of the serum fromthe basal culture medium but shall need a change in the type of theculture medium, that should be a serum-free medium (SFM). Moreover, whenthe avian cell lines necessitate to be grown with growth factors orfeeder cells, the serum weaning is preferably performed after theweaning in growth factors and/or feeder cells.

The feeder cells are animal cells that have been preferably inactivatedby irradiation or chemically treated with mitomycin. The feeder may begenetically modified to express growth factors such as SCF. Preferably,the feeder cells are mouse fibroblasts cell lines such as STO (AmericanType Culture Collection ATCC N^(o)CRL-1503).

The method described above may additionally comprise a step in which thecells obtained in step c) are subjected to a selection or an adaptationin culture media used for large-scale production so as to obtain clonessuitable for the production of vaccines intended for human or animaltherapy.

This process leads to the establishment of new avian embryonic derivedcell lines which are maintained in culture in vitro over a considerableperiod of time. Advantageously, the cells derived from the cell linesobtained in step c) are capable of proliferating for at least 50 days,100 days, 150 days, 300 days or preferably at least 600 days. The 600days do not constitute a time limit because the cell lines obtained arestill alive after much longer time periods. Hence, these lines areconsidered as being able to grow indefinitely in a basic culture mediumfree of exogenous growth factors, serum and/or inactivated feeder layer.The expression “line” is understood to mean any population of cellscapable of proliferating indefinitely in culture in vitro whileretaining to a greater or lesser degree the same morphological andphenotypic characteristics. Of course, the method mentioned above makesit possible to obtain cellular clones derived from cells obtained fromestablished lines. These clones are cells which are geneticallyidentical to the cell from which they are derived by division.

The established cell lines and the cells derived thereof (step c or d)are preferably embryonic derived avian stem cells lines, more preciselythose cells are pluripotent avian embryonic derived stem cells. Theavian embryonic derived stem cells obtainable by the process of theinvention are small, round, individualized cells with a doubling time ofaround 24 hours or less at 39° C. The cells obtainable by the process ofthe invention are at least at passage p60, at least p70, at least p80,at least p90, at least p100, at least p110 at least p120 or at leastp130 or later. The avian embryonic derived stem cells according to theinvention have at least one of the following characteristics:

-   -   a high nucleo-cytoplasmic ratio,    -   an endogenous alkaline phosphatase activity,    -   an endogenous telomerase activity,    -   a reactivity with specific antibodies selected from the group of        antibodies SSEA-1 (TEC01), SSEA-3, and EMA-1.        A doubling time shorter than the doubling time of the avian        cells of step a) of the process of the invention (48 to 72 h at        39° C.), of about 24 hours or less in the same culture        conditions.    -   These cell lines and the cells derived there from are capable of        proliferating for at least 50 days, 100 days, 150 days, 300        days, or preferably at least 600 days in a basal medium, in        particular in a medium such as DMEM, GMEM, HamF12 or McCoy        supplemented with various additives commonly used by persons        skilled in the art. Among the additives, there may be mentioned        non-essential amino acids, vitamins and sodium pyruvate.        However, the cells are able to proliferate in basal medium        without glutamine.    -   These cells lines and the cells derived there from have the        characteristic to grow either as adherent cells or as suspension        cells.        Preferably, the cells of the invention have all the above        mentioned characteristics.

The avian established cell lines of the invention and the cells derivedthereof are useful for the production of biologics such as recombinantpeptides and proteins (i.e antibodies, hormones, cytokines . . . ),viruses, viral vectors, viral particles and viral vaccines.

More precisely, the avian established cell lines of the invention andthe cells derived thereof are useful for the replication of virusesand/or related vectors and particles for the production of live orattenuated, recombinant or not, vaccines against diseases, such cancersand infectious diseases. The viruses, the related viral vectors, theviral particles and viral vaccines are preferably chosen among the groupof adenoviruses, hepadnaviruses, herpes viruses, orthomyxoviruses,papovaviruses, paramyxoviruses, picornaviruses, poxviruses, reovirusesand retroviruses. In a preferred embodiment, the viruses, the relatedviral vectors, viral particles and viral vaccines belong to the familyof poxviruses, and more preferably to the chordopoxviridae. Morepreferably, the virus or the related viral vectors, viral particles andviral vaccines is an avipoxvirus selected among fowlpox virus, canarypox virus (i.e ALVAC), juncopox virus, mynahpox virus, pigeonpox virus,psittacinepox virus, quailpoxvirus, sparrowpoxvirus, starling poxvirus,turkey poxvirus. According to another preferred embodiment, the virus isvaccinia virus.

In another embodiment, the viruses, the related viral vectors, the viralparticles and vaccines belong to the family of orthomyxoviruses, inparticular influenza virus and to the family of paramyxoviruses, inparticular measles, mumps and rubella viruses.

The invention also relates to the biologics, in particular the proteinsand the vaccines, expressed and/or produced in the avian establishedcell lines of the invention.

In a preferred embodiment, the invention is related to the use of theadherent or non-adherent avian established cell lines of the invention,that are genetically, biologically or chemically unmodified, capable ofproliferating indefinitely in culture, and having the abovecharacteristics to replicate live or attenuated viruses of theorthopoxvirus family, more particularly live or attenuated vacciniavirus and recombinant vaccinia viruses.

The invention is aimed at the use of the adherent or non-adherent cellsas defined above to produce live or attenuated vaccines comprisingculturing the adherent or non adherent cell lines established in step c)or d) according to the process described above, inoculating said cellswith viral particles and culturing said cells in a basal medium asmentioned above until cell lysis occurs and, recovering the newlyproduced viral particles released in said medium. The invention isparticularly useful for the production of attenuated virus belonging tothe family of orthopoxvirus, in particular vaccinia virus,Lister-Elstree vaccinia virus strain, modified vaccinia virus such asModified Vaccinia virus Ankara (MVA) which can be obtained from ATCC(ATCC Number VR-1508), NYVAC (Tartaglia et al., 1992 Virology 188:217-232), LC16 m8 (Sugimoto et Yamanouchi 1994 Vaccine 12:675-681),CVI78 (Kempe et al., 1968 Pediatrics 42: 980-985) and other recombinantvaccinia virus. Advantageously, the cells derived from established linesare infected in order to produce a live vaccinia virus or an attenuatedvirus which is a modified vaccinia virus and/or recombinant vaccinia.Said cells may be infected by any technique accessible to personsskilled in the art.

Alternatively, the cells derived from established lines are transfectedor modified in order to produce a live vaccinia virus or an attenuatedvirus which is a modified vaccinia virus and/or recombinant vaccinia.Said cells may be modified by any technique accessible to personsskilled in the art, in particular by non homologous or homologous,directed and/or conditional recombination (Cre-Lox or FLP-FRT system),by transformation with any vector, plasmid, viruses or recombinantviruses in particular with the aid of retroviruses or recombinantretroviruses.

In one particular embodiment, the invention is directed to a method toproduce live or attenuated vaccines such as a vaccine against smallpoxcomprising culturing the adherent or non adherent cell lines establishedin step c) or d) according to the process described above, inoculatingsaid cells with viral particles and culturing said cells in a basalmedium as mentioned above until cell lysis occurs and, recovering thenewly produced viral particles released in said medium. The invention isparticularly useful for the production of attenuated virus belonging tothe family of poxvirus, in particular vaccinia virus, Lister-Elstreevaccinia virus strain, modified vaccinia virus such as Modified Vacciniavirus Ankara (MVA) which can be obtained from ATCC (ATCC NumberVR-1508), NYVAC (Tartaglia et al., 1992 Virology 188: 217-232), LC16 m8(Sugimoto et Yamanouchi 1994 Vaccine 12:675-681), CVI78 (Kempe et al.,1968 Pediatrics 42: 980-985) and others recombinant vaccinia viruses.For example, one can use MVA such as a vaccine against smallpox.

In a second particular embodiment, the invention is directed to a methodto produce live or attenuated vaccines such as a vaccine againstdiseases, more preferably, acquired or infectious diseases; said methodis comprising culturing the adherent or non adherent cell linesestablished in step c) or d) according to the process described above,inoculating said cells with viral recombinant particles and culturingsaid cells in a basal medium as mentioned above until cell lysis occursand, recovering the newly produced viral recombinant particles releasedin said medium. For example, one can use recombinant MVA to expressantigen against:

-   -   acquired diseases such as, for example and without limitation,        prostate cancer, pancreatic cancer, colorectal cancer, lung        cancer, breast cancer, melanoma;    -   infectious diseases such as, for example and without limitation,        AIDS (HIV virus), hepatitis A, hepatitis B, hepatitis C,        malaria, rabies, yellow fever, Japanese encephalitis, mumps,        measles, rubella.        The vaccines produced by the above method are part of the        present invention.        For the remainder of the description, reference will be made to        the legend to the figures below.

LEGEND OF FIGURES

FIG. 1: Growth curve for one cell line of the invention showing the longterm replication of cells.

FIG. 2: population doubling times of S86N45 (EB45) (adherent) and EB14(suspension) cells.

FIG. 3: influence of temperature on S86N45 (EB45) cells growth kinetics

FIG. 4: Growth curve for one cell line of the invention showing the longterm replication of cells with withdrawal of serum (up to 2% of serum).

FIG. 5: Adaptation of S86N45 (EB45) and EB14 cells to growth inserum-free medium (SFM).

FIG. 6: Culture of EB14 suspension cells in a 2 L bioreactor inserum-free medium.

FIG. 7: Growth curve for one cell line of the invention (S86N16) showingthe long term replication of cells with withdrawal of feeder layer.

FIG. 8: Photograph showing the characteristic morphology of avian stemcells N: nucleus, n: nucleolus and C: cytoplasm (isolate S86N99, ×40magnification, photograph taken with a Sony Cyber-shot digital camera)

FIG. 9: Photographs showing the alkaline phosphatase activity of avianstem cell lines which are adherent or which are in suspension Afterfixing (0.1% formaldehyde/0.5% glutaraldehyde, 30 minutes at 4° C.), thecells are rinsed twice in 1×PBS and incubated for between 10 and 30minutes at 37° C. in an NBT/BCIP (Nitro Blue Tetrazolium chloride 0.375mg/ml, 5-bromo-4-chloro-3-indolyl phosphate 0.188 mg/ml, 0.1M Tris pH9.5, 0.05M MgCl₂, 0.1M Nacl) solution. The reaction is stopped by two1×PBS washes and the photographs are taken. A—illustrates thecharacteristic violet coloration of the endogenous alkaline phosphataseactivity obtained with the adherent line S86N45 p87, a line culturedwith no feeder or factor (×40 magnification, Sony Cyber-shot digitalcamera). B—illustrates the violet coloration characteristic of theendogenous alkaline phosphatase activity obtained with the EB14 linemaintained from 8 passages in suspension, line derived from the S86N45cells, cultured in suspension with no feeder or factor (×20magnification, Sony Cyber-shot digital camera). C—S86N45 (EB45)cell-specific markers.

FIG. 10: Viral susceptibility of CEF and adherent S86N45 (EB45) cells(72 hours post-infection—MOI 0.1)

FIG. 11: Viral susceptibility of CEF and adherent S86N45 (EB45) cells atvarious multiplicity of infection (MOI) (48 hours post infection)

FIG. 12: kinetics of MVA-GFP propagation on adherent S86N45 (EB45)cells.

FIG. 13: kinetics of MVA-GFP propagation on suspension EB14 cells.

FIG. 14 MVA-GFP replication on S86N45 (EB45) cells grown on DMEM-F12medium.

FIG. 15: Replication of a wild type MVA virus on S86N45 (EB45) cellsgrown on DMEM-F12 medium (MOI: 0.1)

FIG. 16: MVA replication on suspension EB14 cells on a serum-containingmedium (MOI: 0.2)

FIG. 17: MVA replication on suspension EB14 cells grown in serum-freemedium (MOI: 0.01)

FIG. 18: MVA yields on suspension EB14 cells grown in serum-free medium(MOI: 0.01)

EXAMPLES Example 1 Production and Establishment of the Adherent Cells

The eggs are opened, the yolk is separated from the egg white during theopening. The embryos are removed from the yolk either directly or withthe aid of a Pasteur pipette, or with the aid of a small absorbentfilter paper (Whatmann 3M paper), cut out beforehand in the form of aperforated ring with the aid of a punch. The diameter of the perforationis about 5 mm. These small rings are sterilized using dry heat for about30 minutes in an oven. This small paper ring is deposited on the surfaceof the yolk and centered on the embryo which is thus surrounded by thepaper ring. The latter is then cut out with the aid of small pairs ofscissors and the whole removed is placed in a Petri dish, filled withPBS or with a physiological saline. The embryo thus carried away by thering is cleaned of the excess yolk in the medium and the embryonic disk,thus freed of the excess vitellin, is collected with a Pasteur pipette.

In both cases, the embryos are placed in a tube containing physiologicalmedium (1×PBS, Tris Glucose, medium, and the like). The embryos are thenmechanically dissociated and inoculated on a “feeder” into definedculture medium. Among the preferred conditions used for the culturing,preference is given to the culture medium composed of MacCoy or DF12medium as basal medium supplemented with fetal calf serum at an initialconcentration of 12 to 8%, with nonessential amino acids at 1%, with amixture of vitamins of commercial origin at 1%, with sodium pyruvate ata final concentration of 1 mM, with beta-mercaptoethanol at a finalconcentration of 0.2 mM, glutamine at a final concentration of 2.9 mM,with an initial mixture of antibiotics containing gentamycin at a finalconcentration of 10 ng/ml, penicillin at a final concentration of 100U/ml and streptomycin at a final concentration of 100 μg/ml. Rapidlyafter the first passages of the cells, the mixture of antibiotics is nolonger added to the medium. The expression rapidly is understood to meanafter the first 3 to 5 passages in general. A mixture of nucleosides mayalso be added, this mixture being prepared as described above (Pain etal., 1996). Among the basal media tested under these same conditions andwhich give similar results are the HamF12, Glasgow MEM and DMEM media,the latter supplemented with biotin at a final concentration of 8 mg/l.By way of comparison, the biotin concentration is 0.2 mg/l in the MacCoymedium, 0.0073 mg/l in the HamF12 and 0 in the commercial DMEM and GMEMmedia.

The growth factors and the cytokines added to the culture medium arepreferably factors and cytokines which are recombinant, including mouseSCF at a final concentration of 1 ng/ml, IGF-1 at a final concentrationof 1 to 5 ng/ml, CNTF at a final concentration of 1 ng/ml, IL-6 at afinal concentration of 1 ng/ml, and the soluble IL-6 receptor at a finalconcentration of 0.5 ng/ml to 1 ng/ml. In some experiments, some otherfactors may be added during the first passages. For example up topassage 3 or 10, it is possible to add bFGF to the medium at a finalconcentration of 1 ng/ml and IL-11 at a final concentration of 1 ng/ml.

The inoculation is carried out into this medium on the inactivated“feeder” composed of mouse fibroblasts established as lines, the STOcells. In some cases, these cells were transfected with simpleexpression vectors allowing the expression of growth factors such asavian SCF, constitutively in the STO cells. Thus, this “feeder” producesthe factor in a form which is soluble and/or attached in the plasmamembrane of the cells.

After initial inoculation of the cells directly into this medium, freshmedium can be added or the medium can be partially changed the next day,and then partially or completely during subsequent days, depending onthe rate of adhesion observed for the primary cells. After about 4 to 7days depending on the cases, the initial culture is dissociated andtransferred into new dishes in the same initial medium on theinactivated feeder. After three to five passages, the cells are culturedon an inactivated feeder of STO cells which are non-transfected ortransfected with an expression vector encoding a resistance to anantibiotic such as the gene for resistance to neomycin, to hygromycin,to puromycin and the like. After about twenty passages, the cells areprogressively deprived of growth factors and cytokines. The expression“gradual withdrawal” is understood to mean a removal growth factor bygrowth factor, or group of growth factors by group of growth factors,from the culture medium. In the first embodiment, at one passage, SCF isfirst of all removed, and then, two or three passages later, anothergrowth factor such as IGF-1 for example. If the cells do not exhibitmorphological alterations or a variation in their average rate ofproliferation, the other factors, such as CNTF and IL-6, are thenremoved. In a second preferred embodiment, the withdrawal of growthfactors is performed group of growth factors by group of growth factors.A first group of growth factors composed by SCF, IL6, IL6R and IL11 isremoved then the second group composed of IGF1 and CNTF. In a thirdembodiment, this withdrawal may also be drastic. All the factors are inthis case removed all at once. The cells are then observed and are onlypassaged several days later if their rate of proliferation is modified.The latter solution is generally that which is practiced.

Various isolates are thus obtained and maintained for very long periodsof time. The expression very long periods of time is understood to meanperiods of the order of several weeks with a minimum of 50 days,preferably periods greater than 200 to 400 days, without limitation intime. Periods greater than 600 days are observed.

Regardless of the support used, all the cells which are adherent aredissociated with a proteolytic dissociation enzyme, such as pronase,collagenase, dispase, trypsin, and the like. Preferably, a proteolyticenzyme of bacterial origin is used in order to avoid any potentialcontaminant of animal origin. These cells have the characteristics ofembryonic stem cells with a specific morphology illustrated, by way ofexample, by the photograph of FIG. 8 i.e. a small size, a largenucleo-cytoplasmic ratio, a nucleus with at least one nucleolus which isclearly visible and a very small cytoplasm. These cells arecharacterized by growth in the form of more or less compact solidmasses. The adherent and non-adherent cells exhibit cross-reactivitywith a number of antibodies, as described above in Pain et al. (1996)and in patents U.S. Pat. No. 6,114,168 and EP787,180. The endogenoustelomerase activity component is also present and is an important factorin the “stem” nature of these cells.

Cells of different isolates are obtained and maintained for long periodsof time. Table 1 illustrates a few of the characteristics of theseisolates.

TABLE 1 Name Species Start Stoppage Days Passage Generation S86N16Chicken S86N 26-01-2000 05-08-2001 559 207 692 WL3 Chicken WL 28-06-200009-08-2001 403 153 333 Valo4 Chicken Valo 26-09-2000 07-02-2002 401 135317 S86N45 Chicken S86N 29-01-2001 12-11-2001 287 118 329It will be noted that the term “stoppage” does not correspond to the endof the proliferation of the cells but to a deliberate stoppage of thecell cultures by the experimenter. The number of generation n isobtained by the formula X=2^(n) or X is the theoretical cumulativenumber of cells. This number is available since the cells are counted ateach passage and during each inoculation. The complete history of theculture is thus available. S86N45 cells also named EB45.

Example 2 Passage of the Cells

One of the characteristics of stem cells, in particular somatic stemcells and embryonic stem cells, is their capacity to proliferate invitro for considerable periods of time. In order to propagate and topassage the cells, the culture medium is changed and replaced with freshmedium a few hours before their passage. The curve presented in FIG. 1illustrates a profile of cell growth and establishment.

Example 3 Doubling Time and Average Division Time

3.1 Starting with the established cells in culture and the cellspresented in the preceding examples, a mean division time can becalculated. For all the independent isolates obtained, the rate ofproliferation increases slightly during successive passages, thuscausing the average division time during the establishment of the cellsto vary. In the adherent phase, the cells are initially inoculated on aninactivated feeder layer and are passaged regularly at a constantinitial inoculation density of 1 to 2×10⁶ cells per 100 mm dish (55 cm²dish). Table 2 illustrates the doubling time (d) and the mean divisiontime (MDT in hour) for 3 established cell types as a function of theculture time. It is observed that the mean doubling time decreasesduring the establishment.

TABLE 2 Cells/days 50 100 150 200 250 300 350 400 450 500 550 S86N16 (d)0.30 0.63 1.00 0.86 1.13 1.15 1.47 1.70 1.94 1.50 1.9 S86N16 (MDT) 80 3824 27.9 21.2 20.9 16.3 14.1 12.4 16 12.6 S86N45 (d) 0.49 0.89 0.89 1.452.15 x x x x x x S86N45 (MDT) 49 26.8 27 16.5 11.1 x x x x x x Valo4 (d)0.03 0.61 1.00 1.17 1.26 1.03* 1.08* 1.25* x x x Valo4 (MDT) >48 39.3 2420.5 19 23.3 22.2 19.2 x x x

The mean doubling time d is established for the period of time indicatedin days with the following formula: d=(1/Log 2×(Log X2/X1))×1/(T2−T1)where X2 and X1 are total numbers of cells at the times T2 and T1. Thisformula is the direct consequence of the calculation of the number ofgenerations N by the formula X=2^(n) presented in example 1. The meandivision time (MDT) is then obtained in hours by dividing 24 hours by d.

*The Valo cells are passaged during this establishment on a plasticsupport without the presence of a feeder. The doubling time decreasesand then increases again, when the cells become rehabituated to this newenvironment.

3.2—Chickens have a body temperature of 39° C. Analysis of S86N45 (EB45)and EB14 cells cell growth kinetics was thus initially performed at 39°C. Under these conditions cells were characterized by a very shortgeneration time usually comprised between 15 to 20 hours (FIG. 2).

Example 4 Cell Culture Temperature

The very rapid cycling of S86N45 (EB45) and EB14 cells at 39° C. may besub-optimal for efficient MVA virus production. Cell growth at 37° C.and 35° C. was therefore also analyzed (FIG. 3). As expected, cellcycling is reduced at 37° C. Such conditions should in principle be moreadequate for virus propagation and will thus be selected in the MVAexperiments described below. It is relevant to note that S86N45 (EB45)and EB14 cells can also be grown at 35° C., albeit with a much reducedkinetics. Adaptation of S86N45 and EB14 cells to low temperature (35° C.and even 33° C.) is particularly useful for the production of liveattenuated thermo-sensitive viral vaccines.

Example 5 Control of the Level of Serum for the Proliferation of theLines

5.1—Medium with Low-serum Concentration

During the obtaining of these lines, the culture media used areconventional culture media comprising a base (DMEM, GMEM, HamF12, McCoy,and the like) supplemented with various additives such as non-essentialamino acids, vitamins, and sodium pyruvate. This complex mediumcomprises fetal calf serum, which remains a central component of theculture, even though components of different origins, including plantcomponents, can be gradually used. A process for controlling andhabituating the cells to relatively low proportions of fetal calf serumis presented. It is thus possible to maintain cells in highproliferation (division time >1) with low percentages of serum (2% forexample in the case of the S86N16 cells).

The curves presented in FIG. 4 illustrates the relative reduction ofserum for a given cell type: S86N16 cells, The doubling time and themean division times were also calculated and presented in table 3. Itwill be noted that the mean division time increases as a function of therelative reduction in serum. A recovery phase is nevertheless observedafter some time in culture under the conditions mentioned. This timeremains nevertheless less than 24 h (d>1), which already represents avery advantageous proliferation in industrial terms even at serumconcentrations of 2%, which is already relatively low. Improvements withregard to the different metabolites to be used may be envisaged in orderto increase this time and still further optimize the culture conditions.

TABLE 3 Doubling time and mean division time for S86N16 cells Condition10% 7.5% 3.75% 2% d 2.02 1.51 1.47 1.08 MDT 11.9 15.8 16.3 22.2The examples are taken between passages p204 and p179 for the 10%condition, between p198 and p176 for the 7.5%, between p224 and p201 forthe 3.75% and between p216 and p199 for the 2%.5.2—Adaptation to Serum-free Medium & Growth in Bioreactors

A major further improvement was achieved by the adaptation of S86N45(EB45) and EB14 cells to serum-free medium. Several formulations havebeen tested and a couple of serum-free medium formulations have beenidentified that allow the efficient growth of S86N45 (EB45) and EB14cells (FIG. 5).

In addition, the culture of EB14 cells in serum-containing andserum-free media could be further up-scaled since efficient growth wasreproducibly demonstrated in 2 L bioreactors (FIG. 6). In addition, EB14cells can also be efficiently grown in 3L stirred-tank bioreactors andreach densities over 2 millions cells/ml.

Example 6 Deprivation of the Cells of Feeder Layer

Under the initial culture conditions, the presence of a layer ofinactivated cells appears to be necessary in order to obtain embryonicstem cells as was described above. This feeder layer no longer appearsto be necessary after a number of passages. Only the “culture treated”plastic appears to be important. Indeed, one of the characteristics ofsome eukaryotic cells is to proliferate in adherent form. In order tofacilitate the adhesion of the cells, the various plastic materials usedare “culture” treated. They undergo during their manufacture a treatmentwhich adds charges at the surface of the plastic, which charges promotethe adhesions of the extracellular matrix of the cells. By contrast, thecell culture untreated plastic, often called plastic of bacteriologicalquality, is not surface treated by addition of specific feeders. Theadhesion of the cells thereto is generally very difficult, or evenimpossible, or then induces changes in morphology, and in behavior whichare often drastic. This distinction between the two plastic qualitiesmakes it possible to obtain, depending on the inoculations which arecarried out therein, cells with different behaviors. Gradual deprivationof the cultures of inactivated “feeder” makes it possible to obtain,after a few passages, homogeneous cultures of stem cells directlyinoculated on “culture treated” plastic.

The comparative growth curves for the cells maintained in the presenceand in the absence of inactivated “feeder” are presented with the caseof the S86N16 cells in FIG. 7. This adaptation of the cells isprogressive so as not to lose the stem cell character of the cellsinitially maintained on a “feeder”. Progressive derivatives are thusmade. The obtaining of cells which proliferate on plastic is theaccomplishment of the withdrawal process. In table 4, the division timesshow sensitivity of the cells to their environment. As in the case ofthe progressive withdrawal of serum, an adaptation is obtained with arecovering effect on the cells after a few passages under the conditionsdefined.

TABLE 4 Condition 1.2 0.5 0.3 plastic d 1.95 1.84 1.39 1.42 MDT 12.3 1317.3 16.9The examples are taken between the passages p154 and p131 for the 3conditions 1.2×10⁶, 0.5×10⁶ and 0.3×10⁶ feeder cells and between p161and p139 for the condition on plastic alone.

Example 7 Deprivation of the Cells in Growth Factors

Under the initial culture conditions, the presence of growth factors isnecessary. It is possible to schematically distinguish two families offactors: the cytokines and the trophic factors.

The cytokines are mainly cytokines whose action is through a receptorwhich is associated with the gp130 protein. Thus, LIF, interleukin 11,interleukin 6, CNTF, oncostatin and cardiotrophin have a similar mode ofaction with the recruitment at the level of the receptor of a specificchain and the combination of the latter with the gp130 protein inmonomeric or sometimes heterodimeric form. In a few cases, thecombination of a soluble form of the receptors, a form described interalia for the receptors for interleukin 6 and CNTF, makes it possible toincrease the proliferative effect observed. It has been previously shownthat the addition of at least one of these cytokines appeared to benecessary for obtaining embryonic stem cells.

The trophic factors are mainly SCF, IGF-1 and bFGF, which are also usedat the start of the culture, as described above. Their presence is alsonecessary for obtaining and amplifying the cells.

By progressively reducing these growth factors, it is possible toobtain, after a few passages, culture conditions which allow theproliferation of the embryonic or somatic stem cells without theaddition of an exogenous growth factor. The different markers used tocharacterize these cells are always positive for the cells maintainedwith no factors.

Example 8 Comparison of the Media Used

Inoculated into different media, the cells are not obtained with thesame frequencies. Comparison of the compositions of the media makes theidentification of one of the components in particular difficult. Itappears more likely that the whole combination allows an improvement inthe physiology of the cells. Among the preferred media, the Ham F12medium, the MacCoy medium, the DMEM medium, DMEM-F12 medium and a DMEMmedium enriched with biotin will be noted. Starting with such anisolate, adaptation trials are carried out in these different media.

Example 9 Establishment of the Non-adherent Cells

During the successive passages of the stem cells, a high-densityinoculation directly into the bacteriological dish makes it possible toobtain, after a few passages, embryonic cells which become detached fromtheir substrate and which proliferate in suspension in the form of smallregular aggregates. This proliferation is encouraged over severalpassages by more dilution, mechanical dissociation and non-use ofproteolytic enzyme. The stirring of the cultures is generally carriedout but does not represent a distinguishing factor for obtaining nonadherent cells. Like the adherent cells, these cells have acharacteristic morphology of stem cells, i.e. a small size, a largenucleo-cytoplasmic ratio, a nucleus with at least one nucleolus which isclearly visible and a small cytoplasm. These cells are characterized bya growth in small aggregates which are more or less compact (FIG. 8).These non adherent cells exhibit cross-reactivity with a number ofantibodies, as described above in Pain et al. (1996). These cells arealso positive for the endogenous telomerase activity (as presented inexample 10 for the EB1, EB4 and EB5 cells). In a non adherent phase, thecells exhibit a high proliferation in different media. The initialinoculation density and the very regular supply of fresh medium provideshigh densities which may range above 1×10⁶ cells per ml. Table 5summarizes the main characteristics of a few isolates (parental cells,initial passage of the making into a suspension, number of daysmaintained in culture in suspension, number of passages and ofgenerations obtained before voluntary stoppage of the maintenances). Itcan thus be noted that the passage for the making into a suspension canvary from one isolate to another (see isolate EB1 and EB14) and theproliferation rate (see isolate EB3 and EB14).

TABLE 5 Parental Initial Name cells passage Start Days PassagesGenerations EB1 S86N16 p111 20-01-2001 184 41 120 EB3 S86N16 p11823-01-2001 381 17 40 EB4 S86N45 p100 25-09-2001 44 17 40 EB5 S86N45 p10025-09-2001 44 17 40 EB14 S86N45 p81 05-09-2002 70 24 65

It will be noted that the term “start” corresponds to the cells beingplaced under non-adherence.

We also found that the obtention of non adherent cells is possible afterseveral passages, at any moment, from adherent stem cells thatproliferate with or without feeder layer.

Example 10 Characterization of the Established Cells

The stem cells maintained for long culture times are characterized withthe same criteria as those described above (Pain et al., 1996). It isthus possible to regularly detect the endogenous alkaline phosphataseactivity, illustrated by the photograph of FIGS. 9 a-9 b, the endogenoustelomerase activity (FIG. 9 c) and reactivity with specific antibodiessuch as the antibodies SSEA-1 (TEC-01) and EMA-1.

One of the important criteria during the establishment of the cells isthe presence of telomerase. Various tests were carried out during themaintenance of the cells in culture using a TRAP detection kit(Telomerase PCR Elisa, Roche). The cells are detected positive aftervarious passages in culture. Thus, the telomerase activity is detectablefor the S86N16 cells, the S86N45 (EB45) cells and for the EB1, EB4 andEB5 cells which are derived therefrom in a non adherent form (see table6). The CEFs (Chicken Embryonic Fibroblasts) maintained in primaryculture are considered as negative. The threshold of an OD<0.2 is thethreshold recommended by the kit as the negative threshold. All theanalyses were carried out on an equivalent of 2000 cells.

TABLE 6 Assay of the telomerase activity in various lines at variouspassages Cells Passage Telomerase OD S86N16 p12 1.7 p29 2.8 p185 0.97p204 0.95 S86N16 EB1 p134 1.1 S86N45 (EB45) p50 0.87 p58 1.1 p66 0.96p94 1.2 EB4 p112 1.4 EB5 p112 0.94 CEF* p4 0.07 *CEF: Chicken EmbryonicFibroblast

Of particular importance, the cells of the invention have conserved someessential “stem cell” features. They express a series of stemcell-specific markers known to be present in mouse, chicken and humanembryonic stem cells (eg. Alkaline phosphatase, SSEA-1, EMA-1,telomerase) (FIG. 9). As expected, expression of these markers is lostupon experimental induction of cell differentiation by addition ofretinoic acid (RA) or DMSO (Table 7 and FIG. 9 c). They replicateindefinitely in vitro (FIG. 1); several candidate cell lines have beencultured for more than a year without specific hurdles, such asdifferentiation.

TABLE 7 ES cell-specific markers: “markers expression is decreased upondifferentiation with retinoic acid” WITHOUT MARKERS RETINOIC ACID WITHRETINOIC ACID Alcaline Phosphatase ++++ − SSEA-1 90 <10 EMA-1 90 10Telomerase Activity (OD) >1.5 <0.2The markers SSEA1 and EMA1 are expressed in percentage of labelledcells.

Example 11 Transfection and Induction of the Cells

The stem cells maintained in a growth over the long term are transfectedwith various expression plasmids. It has been shown that avian stemcells could be transfected (Pain et al., 1996). In particular, the nonadherent cells are transfected and various sorting systems make itpossible to identify the stably transfected cells (cell sorting,limiting dilution, and the like). These genetic modifications can bemade at the undifferentiated stage of the stem cell. Once thismodification has been obtained, the cell is then induced todifferentiate spontaneously or by addition of a differentiation inducer.In this case, it is possible to use retinoic acid at concentrations of10⁻⁸ M to 10⁻⁶ M, or dimethyl sulfoxide at concentrations of 1 to 2%final or sodium butyrate at concentrations of 10⁻⁴ to 10⁻⁸ M, or phorbolester (TPA, PMA, and the like) or lipopolysaccharides (LPS) atconcentrations of 1 to 5 μg/ml final. In another example, the cells canform embryoid bodies in suspension, which embryoid bodies can be causedto adhere to plastic after dissociation or nondissociation of the cellsconstituting them. These differentiated cells then proliferate but havea more limited capacity for proliferation over the long term. Bytargeting the genetic modification on a gene which influences theproliferation of the cells, it is possible to make these differentiatedcells capable of proliferating over the long term.

Example 12 Protocol for Infecting a Non Adherent Avian Cell Line (EB1)with a Virus

Amplification of the Cells:

The EB1 or EB14 cells are inoculated into a medium, preferably MacCoy's5A, HAMF12 or DMEM medium, or any other medium of interest, containing5% serum at a concentration of 0.2×10⁶ cells/ml for an initial volume of50 ml in general. They are maintained in culture at 39° C. and at 7.5%CO₂, with stirring. Fresh medium is added every day for the 3 to 4 daysfor which the amplification lasts in order to reach a cell concentrationof 1 to 3×10⁶ cells/ml for a final culture volume of 100 to 250 ml.

The cells in suspension are collected and centrifuged for 10 min at 1000 rpm approximately. The pellet is resuspended in 20 to 50 ml of 1×PBS(Phosphate buffer Salt). The cells are then counted, centrifuged and thepelleted cells are taken up in a serum-free medium at a finalconcentration of 3 to 5×10⁶ cells/ml. Several tubes are then preparedunder these conditions containing 3 to 5×10⁶ cells per tube.

Preparation of the Virus and Infection:

The viral stock having a known titer is rapidly thawed at 37° C. anddiluted in serum-free medium at a titer of 10× to 1000× theconcentration necessary for the final infection. The cells are infectedwith the virus of interest at an m.o.i. (multiplicity of infection) of0.01 to 0.5 according to the types of virus, which involves addingbetween 0.1 and 10% volume/volume of viral suspension to the cellularpellet. After incubating for 1 hour at an optimum temperature for thevirus, in general from 33 to 37° C., the cells are again centrifuged andthe medium removed with care. This step is found to be often necessaryin order to limit the effect of the initial virus in the subsequentprocess. One of the possibilities is to directly dilute the cellswithout centrifuging them again with serum-containing medium (5% ofserum) at a final concentration of 0.2 to 1×10⁶ cells/ml and incubatedagain.

Harvesting of the Supernatant and of the Cells:

After 2 to 4 days of incubation, depending on the viral kinetics and thepotential cytopathic effect of certain viruses, the medium containingthe cells or the cellular debris is harvested.

Depending on the viruses, only the pellet or the supernatant may be ofinterest and contain the viral particles. The cells are harvested andcentrifuged. The collected supernatant is centrifuged again for 5 to 10minutes at 2 500 rpm, and stored at −80° C. before purification of theparticles. An aliquot is collected in order to carry out the titration.The cellular pellet is taken up in 5 ml of serum-free medium, sonicatedand centrifuged for 5 to 10 minutes at 2500 rpm. The supernatantobtained is stored at −80° C. up to the purification and the titrationof an aliquot.

The viral infection and production efficiencies are compared between thevarious conditions performed. For the viruses with cytopathic effects,the titrations are in general carried out by the lysis plaque technique.

Example 13 Protocol for Infecting an Adherent Avian Cell Line (S86N45)with a Virus

Preparation of the Cells:

The cells are inoculated 48 hours before the infection into T150 flasksat a concentration of between 0.03 and 0.06×10⁶ cells/cm² in a medium,preferably MacCoy's 5A, HAMF12 or DMEM medium, or any other medium ofinterest, containing 5% serum. They are maintained at 39° C. and 7.5%CO₂.

Infection:

The viral stock having a known titer is rapidly thawed at 37° C. anddiluted in serum-free medium at a titer of 10× to 1 000× theconcentration necessary for the final infection. The cells are infectedwith the virus of interest at an m.o.i. (multiplicity of infection) of0.01 to 0.5 according to the types of virus, which involves addingbetween 0.1 and 10% volume/volume of viral suspension to the cellmonolayer. The infection is generally carried out in a minimum of medium(from 5 to 10 ml for a 75 cm² flask) in a medium containing 0% serum.After incubating for I hour at the optimum temperature for the virus, ingeneral from 33 to 37° C., 20 ml of medium 5% are added to the flasks.In a particular case, the cells can be washed with PBS in order toremove the particles which might be attached to the cells. In the caseof a cytopathic virus, the cells are observed daily after the infectionin order to monitor the appearance of cell lysis, which indicates goodprogress of the infection.

Harvesting of the Supernatant and of the Cells:

After 2 to 4 days of incubation, depending on the viral kinetics and thepotential cytopathic effect of certain viruses, the medium containingthe supernatant, the cells and the cellular debris are harvested.Depending on the viruses, only the pellet or the supernatant may be ofinterest and contain the viral particles. The cells are harvested andcentrifuged. The collected supernatant is centrifuged again for 5 to 10minutes at 2500 rpm, and stored at −80° C. before purification of theparticles. An aliquot is collected in order to carry out the titration.The cellular pellet is taken up in 5 ml of serum-free medium, sonicatedand centrifuged for 5 to 10 minutes at 2500 rpm. The supernatantobtained is stored at −80° C. up to the purification and the titrationof an aliquot.

The viral infection and production efficiencies are compared between thevarious conditions performed. For the viruses with cytopathic effect,the titrations are in general carried out by the lysis plaque technique.

Example 14 Replication of Modified Vaccinia Virus Ankara (MVA) onAdherent and Non-adherent Avian Stem Cells of the EB45 Line and EB14Line

A series of experiments was performed on the EB45 (S86N45) and EB14cells to determine their respective susceptibilities to MVA infection,the kinetics of MVA propagation, and the viral production yields. TheMVA viruses used in these studies were either a recombinant MVA vectorexpressing the reporter GFP protein (MVA-GFP) or a non-recombinant MVAvirus. Freshly prepared chicken embryonic fibroblasts (CEF) wereincluded in all experiments as control cells.

14.1—Safety Consideration

The MVA virus (titer 2.5×10⁷ TCID50/ml in 0.5 ml vials) was receivedunder frozen conditions. For safety reasons, the MVA virus and infectedcells were kept under controlled conditions (−80° C. freezer) and thecontaminated plastic material was placed into hypochloride solution formore than 1 hour and then place into a bag for full and completeautoclave inactivation.

14.2—Virus Production

14.2.1—Adherent S86N45 (EB45) Cells

1×10⁶ adherent cells are seeded in 100 mm dish the day before infection,in 20 mL medium. 24 hours later, the medium is discarded, cells areincubated at 37° C. with the inoculum (2 mL serum-free medium, at amultiplicity of infection of 0.01 or 0.1 TCID/cell). 1 hour later, theinoculum is discarded, and 20 mL of pre-warmed medium is added to thecells, and the incubation is kept at 37° C. in 5% CO₂. For viruspreparation, the infected cells are harvested by scrapper, transferredin a 50 mL Falcon™ tube and spun at 1200 RPM at room temperature. Thesupernatant (extracellular viruses, EV) is collected, and the cellpellet (intracellular viruses, IV) is diluted in 1 or 2 mL of medium. EVand IV samples undergo both three thawing-freezing cycles, and then theyare sonicated. After centrifugation at 2500 rpm for 10 mn at roomtemperature, EV and IV samples are aliquoted and kept at −80° C. untiltitration.

14.2.2—Suspension EB14 Cells

0.4×10⁶/mL EB14 cells are seeded in 40 mL medium (16×10⁶ cells) in 125mL spinner bottles the day before addition of the viral inoculum, at amoi of 0.01 or 0.1 TCID/cell in the medium. After one hour of virusincubation, 80 mL of pre-warmed medium is added. Incubation is kept at37° C. under wished spin conditions and 5% CO₂. Infected cells are thenharvested at various times post-infection, transfered in 50 mL Falcon™tubes and spun at 1200 RPM at room temperature. The supernatant(extracellular viruses, EV) is collected, and the cell pellet(intracellular viruses, IV) is diluted in 5 or 10 mL of medium. EV andIV samples undergo both three thawing-freezing cycles, and then they aresonicated. After centrifugation at 2500 rpm for 10 mn at roomtemperature, EV and IV samples are aliquoted and kept at −80° C. untiltitration.

14.3—Virus Titration

14.3.1—Titration of MVA by the TCID₅₀ End-point Dilution Method

The titration of MVA viruses are done by the TCID₅₀ end-point dilutionmethod on CEF or DF-1 cells. The assay determines that the samplecontains a sufficient dose of infectious virus to produce infection.TCID₅₀ is determined as the dilution that produced cytopathic effect(CPE) in one-half of the cumulative number of cell cultures. One P96flat bottom is needed for one viral sample titer. Briefly, 15000 CEFcells/100 μL are seeded per well. Eight rows of eleven wells are seeded.The eight rows stand for the height serial 10-fold dilutions of theviral sample (i.e. 10⁻² to 10⁻⁹). For each serial dilution, a 1 mL mixis done in serum-free medium, 100 μL of the mix is dispensed in 10corresponding dilution-wells, and the eleventh row is the controlnon-infected well. The P96 plate is incubated at 37° C. in 5% CO₂.Between 5 to 10 days later, the viral titer is calculated by theReed-Muench method by recording the positive CPE wells.

14.4—Results of the Susceptibility to MVA Infection and Titration

14.4.1—The intrinsic susceptibility of EB45 (S86N45) and EB14 cells toMVA infection was first investigated using the recombinant MVA-GFPvector. This specific vector was selected for these studies to simplifythe monitoring and quantification of the infected cells. EBx and CEFcells were thus treated with different multiplicities of infections(moi) and cells were analysed by fluorescence microscopy andfluorocytometry at several days post-infections.

As shown in FIGS. 10 and 11, all adherent EB45 cells that are stillviable at 48 hours post-infection did strongly express the reporter GFPprotein, even when using a moi as low as 0.1 TCID₅₀s/cell. Of note, FIG.10 also illustrates the much smaller size of EB45 and EB14 cells whencompared to CEF cells.

Altogether, these results clearly demonstrate the high susceptibility ofthe adherent EB45 and EB14 cells to MVA infection.

14.4.2—The following table 8 lists results obtained in the variousMVA-GFP infection experiments performed. All samples were titteredtwice.

TABLE 8 Results of the titration Time Experimental Multiplicity ofpost-infection Titration (in conditions infection (MOI) (PI) (in hours)TCID₅₀/ml) S86N45 (EB45) cells 0.01 96 9.57 in DMEM-F12 96 8.57 medium(100 mm 0.1 72 7.5 diameter dish) 72 7.63 EB14 cells in 0.2 48 7.5DMEM-F12 medium 72 7.63 (120 ml spinner flasks) CEF cells in HAM- 0.0196 7.5 F12 medium (100 mm 96 7.71 diameter dish) 0.1 72 7.39 72 7.9114.3—Propagation of MVA on EB14 and EB45 Cells

Propagation of MVA on the suspension EB14 and adherent EB45 cells wasdetermined by a quantitative analysis of the kinetics of MVA-GFPreplication. EB45 cells were grown in dishes in DMEM-F12 medium and wereinfected with a moi of 0.1, while EB14 cells were cultured in 120 mlspinner flasks in DMEM-F12 medium for 24 hours before infection with amoi of 0.2. The percentage of infected cells was then quantified by FACSanalysis at various times post-infection. As illustrated in FIGS. 12 and13, all cells that are still viable do express GFP at 48 hours (EB45) or72 hours (EB 14) post-infection.

14.4—Viral Yields on Adherent EB45 Cells Grown in Serum-containingMedium

14.4.1—The viral productivity of the adherent EB45 cells was analysedusing cells grown in DMEM-F12. MVA was found to be very efficientlyreplicated in EB45 in DMEM-F12 and to achieve yields higher than the oneobtained with control CEF cells (FIG. 14).

14.4.2—In a further series of experiments, a non-recombinant MVA virus(from the ATCC) was used for a comparative replication study on CEFcells and EB45 cells: confirming previous results with the MVA-GFPvector, a higher production yields were again obtained with this MVAvirus (FIG. 15).

Altogether, these results demonstrate the high susceptibility to MVAinfection and the efficient virus production of the adherent EB45 cells,which are higher than in chicken embryonic fibroblasts. In addition, allthese experiments were performed under standard conditions. It istherefore reasonable to argue that even higher virus yields may beachieved upon optimisation of the experimental conditions, and inparticular by using optimal cell culture media.

14.5—Viral Yields on Suspension EB14 Cells Grown on Serum-containingMedium

The viral productivity of EB14 cells has been determined in spinnerflasks using cells grown in DMEM-F12 medium. Results of a first seriesof experiments using a multiplicity of infection of 0.1 are shown inFIG. 16. These data support the previous results obtained with theadherent cells and confirm the ability of the suspension EB14 cells toefficiently produce recombinant MVA viruses at yields close to 100TCID₅₀/Cell, two fold higher than the one obtained with chickenembryonic fibroblasts.

14.6—Viral Yields on Suspension EB14 Cells Grown on Serum-free Medium

Ideally, viral vaccines production should be performed on suspensioncells grown in serum-free medium in bioreactors. In order to investigatethe production of MVA in serum-free media, a series of experiments wasinitiated in which EB14 suspension cells have been infected with theMVA-GFP vector in spinner flasks in serum-free medium at two differentmultiplicities of infection (0.01 & 0.1). FACS analyses of the cellsconfirm the efficient infection of EB14 cells in the two experimentalconditions (FIG. 17). In addition, and as expected, these experimentsshow that infection is more rapid when using an moi of 0.1, while at anmoi of 0.01 cells are viable longer and able to produce virus progenyfor a longer period of time (data not shown).

Analysis of virus yields confirm that efficient MVA production isachieved by the suspension EB14 cells grown in suspension in serum-freeand protein-free medium (FIG. 18). A virus yield higher than the oneunusually obtained with CEF cells is routinely obtained. In addition,analysis of the distribution of the infectious particles indicate thatmost virions are retained within the cells and only a fraction issecreted in the supernatant (FIG. 18).

EB14 and S86N45 cells are well characterized non-genetically engineeredavian embryonic stem cells that can be efficiently grown in serum-freemedium, either in suspension or as adherent cells. The inventorsdemonstrate that the cells are highly susceptible to infection andpropagation of a recombinant and a non-recombinant modified-vacciniavirus Ankara, and results indicate that viral production is at least twoto three fold higher than in control CEF cells. Altogether, thesefeatures make of cells of the invention, mainly EB14 and EB45, a highlypromising cell substrate to replace the current egg-based or CEF-basedproduction system for the production of MVA-based vectors.

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1. A method for replicating a native or recombinant vaccinia viruscomprising the steps of: 1) producing avian embryonic derived stem cellsby: a) culturing avian embryonic cells in a complete culture mediumcomplemented in serum containing: i) exogenous growth factors comprisingthe trophic factors SCF, IGF-1 and bFGF, and cytokines whose action isthrough a receptor which is associated with the gp130 protein, saidcytokines being selected from the group consisting of LIF, interleukin11, interleukin 6, interleukin 6 receptor, CNFT, oncostatin andcardiotrophin; and ii) a feeder layer; b) passage by modifying theculture medium so as to obtain the withdrawal of said growth factors, ofthe serum and/or of the feeder layer; c) establishing adherent ornon-adherent cell lines capable of proliferating in a basal medium inthe absence of exogenous growth factors, serum and/or inactivated feederlayer; 2) inoculating the resultant avian embryonic derived stem cellswith viral particles of said vaccinia virus; and 3) culturing said cellsin a basal medium until cell lysis occurs and newly produced viralparticles of said vaccinia virus are released in said medium.
 2. Themethod according to claim 1, wherein said avian embryonic cells arechicken or duck embryonic cells.
 3. The method according to claim 1,wherein the feeder layer is inactivated.
 4. The method according toclaim 1, wherein the avian embryonic derived stem cells produced instep 1) are non-adherent stem cells which proliferate in suspension in amedium free of exogenous growth factors, serum and feeder cells.
 5. Themethod according to claim 1, wherein said vaccinia virus is the nativeModified Vaccinia virus Ankara (MVA) or a recombinant thereof.
 6. Themethod according to claim 1, wherein the avian embryonic derived stemcells produced in step 1) are capable of proliferating for at least 600days.
 7. The method according to claim 1, wherein the avian embryonicderived cell lines produced in step 1) are non-adherent stem cells whichproliferate in suspension in a medium free of exogenous growth factors.8. The method according to claim 1, wherein the avian embryonic derivedcell lines produced in step 1) are non-adherent stem cells whichproliferate in suspension in a medium free of serum (serum-free medium).9. The method according to claim 1, wherein the avian embryonic derivedcell lines produced in step 1) are non-adherent stem cells whichproliferate in suspension in a medium free of exogenous growth factorsand serum.
 10. The method according claim 1, wherein the avian embryonicderived cell lines produced in step 1) have at least one of thefollowing characteristics: a high nucleo-cytoplasmic ratio, anendogenous alkaline phosphatase activity, an endogenous telomeraseactivity, a reactivity with specific antibodies selected from the groupof antibodies consisting of SSEA-1 (TEC01), SSEA-3 and EMA-1.
 11. Themethod according to claim 1, wherein said basal medium is selected fromthe group consisting of DMEM, GMEM, HamF12 and McCoy basal mediumsupplemented with additives.